MCF-7 cell monolayers, untreated or treated as indicated, were fixed
in 4% PFA (EMS) in 0.25 M HEPES (pH 7.4) for 1 hr at room temperature, then
in 8% PFA in the same buffer overnight at 4oC. Samples were
infiltrated, frozen and sectioned as previously described (Folsch et al., 2001 (link)). The sections were
immunolabeled with the following primary antibodies diluted in PBS/1% fish
skin gelatin. For single stainings, sections were incubated with rabbit
anti-Rab5 antibody (Cell Signaling) at 1/10. For co-staining, sections were
incubated with rabbit anti-TOM20 (Santa Cruz Biotechnology) at 1/10 and
mouse anti-Rab5 (Cell Signaling) at 1/20. The sections were then incubated
with IgG antibodies, followed directly by 10 nm (for rabbit anti-Rab5 or
rabbit anti-TOM20) or 5 nm (mouse anti-Rab5) protein A-gold particles before
examination with a Philips CM120 Electron Microscope (Eindhoven, the
Netherlands) under 80 kV.
in 4% PFA (EMS) in 0.25 M HEPES (pH 7.4) for 1 hr at room temperature, then
in 8% PFA in the same buffer overnight at 4oC. Samples were
infiltrated, frozen and sectioned as previously described (Folsch et al., 2001 (link)). The sections were
immunolabeled with the following primary antibodies diluted in PBS/1% fish
skin gelatin. For single stainings, sections were incubated with rabbit
anti-Rab5 antibody (Cell Signaling) at 1/10. For co-staining, sections were
incubated with rabbit anti-TOM20 (Santa Cruz Biotechnology) at 1/10 and
mouse anti-Rab5 (Cell Signaling) at 1/20. The sections were then incubated
with IgG antibodies, followed directly by 10 nm (for rabbit anti-Rab5 or
rabbit anti-TOM20) or 5 nm (mouse anti-Rab5) protein A-gold particles before
examination with a Philips CM120 Electron Microscope (Eindhoven, the
Netherlands) under 80 kV.